U.S. patent application number 12/245091 was filed with the patent office on 2009-02-05 for device for measuring extracellular potential and method of manufacturing device.
Invention is credited to Fumiaki Emoto, Masaya Nakatani, Hiroaki Oka.
Application Number | 20090035846 12/245091 |
Document ID | / |
Family ID | 29738322 |
Filed Date | 2009-02-05 |
United States Patent
Application |
20090035846 |
Kind Code |
A1 |
Nakatani; Masaya ; et
al. |
February 5, 2009 |
DEVICE FOR MEASURING EXTRACELLULAR POTENTIAL AND METHOD OF
MANUFACTURING DEVICE
Abstract
A device for measuring an extracellular potential of a test cell
includes a substrate having a well formed in a first surface
thereof and a first trap hole formed therein. The well has a
bottom. The first trap hole includes a first opening formed in the
bottom of the well and extending toward a second face of the
substrate, a first hollow section communicating with the first
opening via a first connecting portion, and a second opening
extending reaching the second surface and communicating with the
first hollow section via a second connecting portion. The first
connecting portion has a diameter smaller than a maximum diameter
of the first hollow section, greater than a diameter of the second
connecting portion, and smaller than a diameter of the test cell.
The device can retain the test cell securely and accept chemicals
and the test cell to be put into the device easily.
Inventors: |
Nakatani; Masaya; (Hyogo,
JP) ; Oka; Hiroaki; (Osaka, JP) ; Emoto;
Fumiaki; (Osaka, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK L.L.P.
2033 K. STREET, NW, SUITE 800
WASHINGTON
DC
20006
US
|
Family ID: |
29738322 |
Appl. No.: |
12/245091 |
Filed: |
October 3, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10485644 |
Feb 3, 2004 |
|
|
|
PCT/JP03/06920 |
Jun 2, 2003 |
|
|
|
12245091 |
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Current U.S.
Class: |
435/288.4 |
Current CPC
Class: |
G01N 33/552 20130101;
G01N 33/54353 20130101; G01N 33/48728 20130101 |
Class at
Publication: |
435/288.4 |
International
Class: |
C12M 1/34 20060101
C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 5, 2002 |
JP |
2002-163934 |
Aug 1, 2002 |
JP |
2002-224563 |
Claims
1. A device for measuring an extracellular potential of a test
cell, said device comprising: a substrate including a base, an
intermediate layer stacked on the base, and a plate stacked on the
intermediate layer; and an insulator connected to intermediate
layer, wherein the base and the intermediate layer have a well
formed in the base and the intermediate layer, the well having a
bottom and extending from the base to the intermediate layer, the
insulator is provided on the bottom of the well and covers the
bottom, the plate has a first surface and a second surface opposite
to the first surface, the first surface of the plate being situated
on the intermediate, the plate has a through-hole allowing the well
to communicate with the second surface of the substrate, and the
intermediate layer has a resistivity greater than a resistivity of
the plate, wherein the test cell is arranged to be held at an
opening of the through-hole which opens to the well.
2. The device of claim 1, wherein the through-hole has an opening
which opens to the well, and a diameter of the opening of the
through-hole is smaller than a diameter of the test cell.
3. The device of claim 1, wherein the insulator covers the bottom
of the well entirely.
Description
[0001] This is a continuation application of U.S. application Ser.
No. 10/485,644, filed Feb. 3, 2004, which is the National Stage of
International Application No. PCT/JP03/06920, filed Jun. 2,
2003.
TECHNICAL FIELD
[0002] The present invention relates to a device for evaluating a
biosample, such as a cell, easily and fast by measuring an
extracellular potential an electrochemical change generated by the
biosample. The present invention also relates to a method of
manufacturing the device.
BACKGROUND ART
[0003] Drugs are generally screened according to electrical
activities of the cell as an index by a patch clamp method or a
method using a chemical, such as a fluorochrome or luminescence
indicator. In the patch clamp method, a micro-electrode probe
electrically records an ion transportation through a single channel
of a protein molecule at a micro-section called "patch" of cell
membrane attached to a tip of a micropipet. This method is one of
the few methods that can evaluate functions of a protein molecule
in real time. (Refer to "Molecular Biology of the Cell" third
edition by Garland Publishing Inc. New York. 1994, written by Bruce
Alberts et al. Japanese Edition "Molecular Biology of the Cell"
pages 181-182, published from Kyouikusha Inc. 1995)
[0004] A fluorochrome or luminescence indicator which emits light
in response to a change of a density of a specific ion monitors
migration of the ion in a cell, thereby measuring the electrical
activities of the cell.
[0005] The patch clamp method requires expertise for producing and
operating the micropipet, and requires a long time to measure one
sample, thus not being suitable for screening a large number of
chemical-compound candidates. The method using the fluorochrome or
the like can screen a large number of chemical-compounds candidates
fast, but requires dyeing cells. A background of the cells may be
colored due to pigment in measuring, and is decolorized according
to a lapse of time, thus reducing an S/N ratio.
SUMMARY OF THE INVENTION
[0006] A device for measuring an extracellular potential of a test
cell includes a substrate having a well formed in a first surface
thereof and a first trap hole formed therein. The well has a
bottom. The first trap hole includes a first opening formed in the
bottom of the well and extending toward a second face of the
substrate, a first hollow section communicating with the first
opening via a first connecting portion, and a second opening
extending reaching the second surface and communicating with the
first hollow section via a second connecting portion. The first
connecting portion has a diameter smaller than a maximum diameter
of the first hollow section, greater than a diameter of the second
connecting portion, and smaller than a diameter of the test
cell.
[0007] The device can retain the test cell securely and accept
chemicals and the test cell to be put into the device easily.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a perspective view of a device for measuring an
extracellular potential in accordance with Exemplary Embodiment 1
of the present invention.
[0009] FIG. 2 is a sectional view of the device in accordance with
Embodiment 1.
[0010] FIG. 3 is an enlarged sectional view of the device in
accordance with Embodiment 1.
[0011] FIG. 4 is a sectional view of the device in accordance with
Embodiment 1 for illustrating its usage.
[0012] FIG. 5 is an enlarged sectional view of the device in
accordance with Embodiment 1.
[0013] FIG. 6 is an enlarged sectional view of the device in
accordance with Embodiment 1.
[0014] FIG. 7 is an enlarged sectional view of the device in
accordance with Embodiment 1.
[0015] FIG. 8 is an enlarged sectional view of the device in
accordance with Embodiment 1.
[0016] FIG. 9 is an enlarged sectional view of the device in
accordance with Embodiment 1.
[0017] FIG. 10 is a sectional view of the device in accordance with
Embodiment 1 for illustrating a method of manufacturing the
device.
[0018] FIG. 11 is a sectional view of the device in accordance with
Embodiment. 1 for illustrating the method.
[0019] FIG. 12 is a sectional view of the device in accordance with
Embodiment. 1 for illustrating the method.
[0020] FIG. 13 is an enlarged sectional of the device in accordance
with Embodiment 1 for illustrating the method.
[0021] FIG. 14 is an enlarged sectional of the device in accordance
with Embodiment 1 for illustrating the method.
[0022] FIG. 15 is an enlarged sectional of the device in accordance
with Embodiment 1 for illustrating the method.
[0023] FIG. 16 is an enlarged sectional of the device in accordance
with Embodiment 1 for illustrating the method.
[0024] FIG. 17 is an enlarged sectional of the device in accordance
with Embodiment 1 for illustrating the method.
[0025] FIG. 18 is an enlarged sectional of the device in accordance
with Embodiment 1 for illustrating the method.
[0026] FIG. 19 is an enlarged sectional of the device in accordance
with Embodiment 1 for illustrating the method.
[0027] FIG. 20 is an enlarged sectional view of the device in
accordance with Embodiment 1 for illustrating another method of
manufacturing the device.
[0028] FIG. 21 is an enlarged sectional view of the device in
accordance with Embodiment 1 for illustrating another method.
[0029] FIG. 22 is an enlarged sectional view of the device in
accordance with Embodiment 1 for illustrating another method.
[0030] FIG. 23 is a perspective view of a device for measuring an
extracellular potential in accordance with Exemplary Embodiment 2
of the invention.
[0031] FIG. 24 is a sectional view of the device in accordance with
Embodiment 2.
[0032] FIG. 25 is an enlarged sectional view of the device in
accordance with Embodiment 2.
[0033] FIG. 26 is a sectional view of the device in accordance with
Embodiment 2 for illustrating a method of manufacturing the
device.
[0034] FIG. 27 is a sectional view of the device in accordance with
Embodiment 2 for illustrating the method.
[0035] FIG. 28 is a sectional view of the device in accordance with
Embodiment 2 for illustrating the method.
[0036] FIG. 29 is a sectional view of another device for measuring
an extracellular potential in accordance with Embodiment 1.
[0037] FIG. 30 is a perspective view of the device for measuring an
extracellular potential in accordance with Exemplary Embodiment 3
of the invention.
[0038] FIG. 31A is a sectional view of the device in accordance
with Embodiment 3.
[0039] FIG. 31B is an enlarged sectional view of the device in
accordance with Embodiment 3.
[0040] FIG. 32 is a sectional view of the device in accordance with
Embodiment 3 for illustrating an operation of the device.
[0041] FIG. 33 is an enlarged sectional view of the device in
accordance with Embodiment 3.
[0042] FIG. 34 is an enlarged sectional view of the device in
accordance with Embodiment 3.
[0043] FIG. 35 is a sectional view of the device in accordance with
Embodiment 3 for illustrating a method of manufacturing the
device.
[0044] FIG. 36 is an enlarged sectional view of the device in
accordance with Embodiment 3 for illustrating the method.
[0045] FIG. 37 is an enlarged sectional view of the device in
accordance with Embodiment 3 for illustrating the method.
[0046] FIG. 38 is a sectional view of the device in accordance
Embodiment 3 for illustrating the method.
[0047] FIG. 39 is a sectional view of the device in accordance
Embodiment 3 for illustrating the method.
[0048] FIG. 40 is a sectional view of the device in accordance
Embodiment 3 for illustrating the method.
[0049] FIG. 41 is a sectional view of the device in accordance
Embodiment 3 for illustrating the method.
[0050] FIG. 42 is a sectional view of the device in accordance
Embodiment 3 for illustrating the method.
[0051] FIG. 43 is a sectional view of a device for measuring an
extracellular potential in accordance with Exemplary Embodiment 4
of the invention.
[0052] FIG. 44 is an enlarged sectional view the device in
accordance with Embodiment 4 for illustrating a method of
manufacturing the device.
[0053] FIG. 45 is an enlarged sectional view of the device in
accordance with Embodiment 4 for illustrating the method.
[0054] FIG. 46 is an enlarged sectional view of a device for
measuring an extracellular potential in accordance with Exemplary
Embodiment 5 of the invention for illustrating a method of
manufacturing the device.
[0055] FIG. 47 is an enlarged sectional view of the device in
accordance with Embodiment 5 for illustrating the method.
[0056] FIG. 48 is an enlarged sectional view of the device in
accordance with Embodiment 5 for illustrating the method.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Exemplary Embodiment 1
[0057] FIG. 1 is a perspective view of a device for measuring an
extracellular potential in accordance with Exemplary Embodiment of
the present invention. FIG. 2 is a sectional view of the device.
FIG. 3 is an enlarged sectional view of the device.
[0058] The measuring device includes substrate 1 made of silicon
having well 2 formed therein. Bottom 3 of well 2 has plural
trap-holes 101 formed therein for retaining cells. Each of the
trap-holes 101 includes first opening 4, hollow section 6, and
second opening 5 aligned in this order on a straight line. A
diameter of first opening 4 is smaller than a maximum diameter of
hollow section 6, and greater than a diameter of second opening
5.
[0059] Respective specific sizes of those portions are optimally
determined according to a size of a test cell. For a test cell
having a diameter of 25 .mu.m, for instance, first opening 4 has a
diameter of 20 .mu.m, which is smaller than 25 .mu.m, and hollow
section 6 has the maximum diameter of 35 .mu.m, which is greater
than that of the cell. A diameter of second opening 5 is determined
to be about 10 .mu.m, which is smaller than that of the test cell.
A test cell generally has a diameter ranging from several
micrometers to several tens of micrometers. Therefore, the diameter
of first opening 4 is preferably 10-50 .mu.m, the diameter of
second opening 5 is 1-5 .mu.m, and the maximum diameter of hollow
section 6 is accordingly determined to be an optimal value between
10 .mu.m and 100 .mu.m.
[0060] As shown in FIG. 3, detecting electrode 7 made of gold is
formed at least on an inner wall of second opening 5 and a lower
part of hollow section 6, and leader electrode 8 made of gold is
provided on the lower surface of substrate 1. Electrode 7 is
electrically connected to electrode 8 at second opening 5. No
conductive material is provided on an upper part of hollow section
6, so that detecting electrode 7 is electrically insulated from
well 2.
[0061] Usage of the measuring device will be described below. FIG.
4 is a sectional view of the device having well 2 containing test
cell 9 and culture solution 10 put thereinto. FIG. 5 through FIG. 9
are enlarged sectional views of first opening 4, second opening 5
and hollow section 6.
[0062] As shown in FIGS. 4 and 5, just after putting culture
solution 10 and test cells 9 into well 2, cells 9 float in solution
10. Not only well 2 but also first opening 4, hollow section 6, and
second opening 5 are filled with solution 10, and then, solution
overflows from second opening 5. At this moment, as shown in FIG.
6, floating cell 9 is sucked onto first opening 4 by a pressure of
solution 10 in well 2. If the pressure is small, solution 10 may be
sucked with a suction pump through second opening 5, thus allowing
floating cell 9 to be sucked onto first opening 4 more
securely.
[0063] Since the diameter of first opening 4 is smaller than that
of cell 9, cell 9 receives a resistance when passing through
opening 4, as shown in FIG. 7. However, due to being forced by the
pressure and the suction, cell 9 can reach hollow section 6, while
the cell deforms. As shown in FIG. 8, cell 9 reaching hollow
section 6 still receives the pressure of culture solution 10 from
well 2 even if the suction stops. Since cell 9 has the diameter
greater than that of opening 4, and since first opening 4 is
provided substantially vertically, cell 9 does not return to well 2
as long as an external force which sucks cell 9 is not applied from
well 2. Thus, cell 9 is retained in hollow section 6.
[0064] Hollow section 6 has an oval shape, which has a lateral
diameter greater than a vertical diameter, and the vertical
diameter is smaller than the diameter of cell 9, as shown in FIGS.
7-9. These dimensions allow cell 9 to be held in hollow section 6
without fail. At that moment, chemicals (not shown) are doped into
culture solution 10 in well 2 and permeate into solution 10. The
chemicals activate test cell 9, as shown in FIG. 9, and have the
cell 9 generate an electric signal at second opening 5. The
electric signal changes an electric potential of solution 10 at
second opening 5. This change of the potential is detected by
detecting electrode 7 and leader electrode 8 which both contact
solution 10.
[0065] As such, the measuring device in accordance with Embodiment
1 includes detecting electrode 7 electrically insulated from well
2, and hollow section 6 retains test cell 9 securely. In other
words, culture solution 10 at second opening 5 is electrically
insulated from solution 10 in well 2. Therefore, the electric
signal generated through the activities of the cell does not leak
to solution 10 in well 2, and is detected by detecting electrode 7
provided on second opening 5.
[0066] If any one of the trap holes retains the test cell, an
extracellular potential can be measured.
[0067] First opening 4 may have a tapered shape flaring towards
well 2, as shown in FIG. 29. This shape allows test cell 9 to enter
into opening 4 from well 2 easily. If a diameter of opening 4 at a
boundary of opening 4 against hollow section 6 is smaller than the
maximum diameter of hollow section 6, test cell 9 is prevented from
returning to well 2. Test cell 9 is thus trapped in hollow section
6, and the measuring device has a high retention rate of the cell.
In this device, the diameter of hollow section 6 is greater than
the diameter of first opening 4 at the boundary against hollow
section 6, and the diameter of first opening 4 at the boundary
against hollow section 6 is greater than the diameter of second
opening 5.
[0068] A diameter of first opening 4 at a boundary against well 2
may be smaller than twice the diameter of the test cell, thus
preventing plural cells from entering into opening 4 simultaneously
and from clogging opening 4.
[0069] In the measuring device in accordance with Embodiment 1,
test cell 9 cannot return to well 2 after entering trap hole 101.
Thus, test cell 9, a somatic sample, contaminates well 2 and
trap-hole 101 during the measurement. The measuring device may be
disposable and not re-used, thus preventing cell 9 from being
removed.
[0070] Next, processes for manufacturing the measuring device in
accordance with Embodiment 1 will be described below. FIG. 10
through FIG. 19 are sectional views of the measuring device which
illustrate the processes.
[0071] First, as shown in FIG. 10, resist mask 11 is provided on
silicon substrate 1 by a photo-lithography method in order to form
well 2. Then, as shown in FIG. 11, well 2 is formed by etching
substrate 1 up to a predetermined depth by a wet etching method or
a dry etching method. The wet etching method may employ KOH or
tetramethyl ammonium hydroxide (TMAH) as an etching solution. The
dry etching method may employ SF.sub.6 or CF.sub.4 as an etching
gas.
[0072] Then, as shown in FIG. 12, resist mask 12 for forming first
opening 4 is provided on a bottom of well 2, and resist mask 13 for
forming second opening 5 is provided on a lower surface of silicon
substrate 1. The diameters of openings 4 and 5 are determined
according to a size of test cell 9. The diameter of first opening 4
is greater than that of second opening 5.
[0073] Next, as shown in FIG. 13, substrate 1 is etched from well 2
up to a predetermined depth for forming first opening 4. Substrate
1 may be etched preferably by a dry etching method employing both
etching-accelerator gas and etching-suppressor gas. The accelerator
gas may be SF.sub.6 or CF.sub.4 accelerating an etching of silicon
not only depth wise but also lateral wise. The gas may be mixed
with CHF.sub.3 or C.sub.4F.sub.8, which suppresses the etching, and
forms a protective film made of polymer of CF.sub.2 on the wall of
the opening, thus allowing the substrate to be etched only below
the mask.
[0074] In order to etch the substrate in a vertical direction, the
following steps are repeated. That is, the substrate is etched a
little with the etching-accelerator gas, and then, the protective
film is formed with the etching-suppressor gas. These steps form
the opening substantially vertically. According to an experiment,
first opening 4 having a diameter of 20 .mu.m is formed by the
following steps. SF.sub.6 flows at a rate of 130 sccm to generate
plasma for 13 seconds, thereby etching substrate 1 by 1 .mu.m.
Then, C.sub.4F.sub.8 flows at a rate of 85 sccm to generate plasma
for 7 seconds, thereby forming the protective film having a
thickness of 0.01 .mu.m. The steps of etching substrate 1 and
forming the protective film are repeated about 60 times, thereby
forming a substantially vertical opening having a depth of 60
.mu.m.
[0075] The protective film is formed not only on the wall of first
opening 4 but also on the bottom with the etching-suppressor gas.
The protective film formed on the bottom can be removed by the
etching-accelerator gas more easily than the protective film on the
wall, thus allowing the substrate to be etched only downward.
[0076] First opening 4 is thus formed, while the protective film is
formed with the etching-suppressor gas. After first opening 4 is
formed, the protective film is formed on the wall of opening 4. The
film protects the wall of opening 4 from damage during the forming
of hollow section 6.
[0077] Then, as shown in FIG. 14, substrate 1 is etched from its
lower surface in order to form second opening 5. The
etching-accelerator gas and the etching-suppressor gas are
alternately used similarly to the forming of first opening 4, thus
allowing the wall of second opening 5 to be formed substantially
vertically.
[0078] Further, the protective film is formed with the
etching-suppressor gas, similarly to the forming of first opening
4, to complete the forming of second opening 5. The wall of opening
5 is thus protected by the film securely, thus being prevented from
being damaged when hollow section 6 is formed in later
processes.
[0079] Next, as shown in FIG. 15, substrate 1 is etched from first
opening 4 only with the etching-suppressor gas. The protective film
is provided on the wall of opening 4 at the previous process, thus
allowing the substrate to be etched downward without damage to the
wall. A portion which is newly etched does not have a protective
film thereon, thus being etched also laterally. This etching forms
hollow section 6 which is provided between first opening 4 and
second opening 5 and is wider than first opening 4, as shown in
FIG. 15. An appropriate amount of substrate 1 is etched to have
hollow section 6 shaped in the oval having the lateral diameter
greater than the vertical diameter.
[0080] After hollow section 6 communicates with second opening 5,
the protective film is still formed on the wall of opening 5. The
film protects the wall of opening 6 from damage even though
substrate 1 is being etched for a while until hollow section 6 has
a predetermined size. If the substrate is excessively etched,
hollow section 6 expands not only in a lateral direction but also
in all directions, as denoted by dotted lines shown in FIG. 15. The
substrate is finished appropriately to etch.
[0081] The etching accelerator gas used in the above-described
etching may include SF.sub.6 or CF.sub.4 and, however, preferably
includes XeF.sub.2 which hardly etches the protective film. The gas
of XeF.sub.2 forms hollow section 6 with little damage on the wall.
The gas of XeF.sub.2, however, needs a long time to etch the
protective film on the bottom of the opening formed in the previous
process. In order to overcome this problem, the protective film on
the bottom may be etched with the gas, such as SF.sub.6, CF.sub.4
or Ar, before the gas of XeF.sub.2 is used.
[0082] According to Embodiment 1, first opening 4, second opening 5
and hollow section 6 are formed in this order. However, second
opening 5, first opening 4 and hollow section 6 may be formed in
this order, or first opening 4, hollow section 6 and second opening
5 may formed in this order. Hollow section 6 may be etched from
second opening 5. In this case, the substrate is etched carefully
to allow hollow section 6 to be greater than first opening 4.
[0083] Next, as shown in FIG. 16, all the resist masks are removed,
and then, gold particles 14 are attached onto the wall by a vapor
deposition method, thereby forming detecting electrode 7. In this
process, gold particles 14 are discharged from a target of first
opening 4. Particles 14 discharged from the target run straight,
thus passing through first opening 4. Then, as shown in FIG. 17,
the particles deposit only on an inner wall of opening 4, a lower
part of hollow section 6, and an inner wall of opening 5. In other
words, detecting electrode 7 is formed only on the inner wall of
second opening 5 and the lower part of hollow section 6.
[0084] Then, as shown in FIG. 18, leading electrode 8 made from
gold is formed on a surface of the substrate at second opening 5.
Since second opening 5 has a diameter smaller than that of first
opening 4, gold particles 15 run straight and deposit only on the
inner wall of opening 5 and a portion of the inner wall of opening
4. As shown in FIG. 19, detecting electrode 7 formed on the lower
part of hollow section 6 and the inner wall of second opening 5 is
electrically insulated from the gold provided on the inner wall of
first opening 4. In order to attach the gold securely to substrate
1, a buffer layer of chrome or titan may be provided on substrate
1, and the gold can be attached on the buffer layer. In order to
avoid depositing the gold on the bottom of well 2, the gold is
deposited before resist mask 12, which has been disposed for
forming first opening 4, is removed. The mask prevents the gold
from depositing on the bottom of well 2 after resist mask 12 is
removed. The gold may be deposited by a sputtering instead of the
vapor-deposition.
[0085] The second openings of the trap-holes have conductors formed
on the walls of the openings, and the conductors are
short-circuited with each other at the lower surface below the
well. This structure creates a parallel connection of electric
potential changes around the test cells held in the trap-holes, so
that the change in the electric potential of each test cell may be
detected even if each electric potential change is small.
[0086] The manufacturing method in accordance with Embodiment 1
allows silicon substrate 1 to have well 2 and first opening 4,
second opening 5, and hollow section 6 which retains the test cell
securely, thus providing a reliable device for measuring an
extracellular potential.
[0087] According to Embodiment 1, substrate 1 is made from silicon
and however, may be made of material which can be dry-etched easily
to be etched straight and laterally through switching etching
gases. For instance, glass and quartz can be etched in a depth
direction with gas, such as SF.sub.6 or CF.sub.4, and in a lateral
direction with gas of HF.
[0088] According to Embodiment 1, first opening 4 is provided
substantially perpendicularly to the bottom of well 2. First
opening 4, upon having a tapered shape having a diameter at well 2
greater than that at hollow section 6, may be formed by the
following processes. When the gas including the etching-accelerator
gas and the etching suppressor gas mixed is used, a concentration
of the etching-accelerator gas is reduced according to proceeding
of the etching from well 2 toward hollow section 6. This operation
allows the wall of opening 4 to taper, as shown in FIG. 20. This
tapered shape allows test cell 9 to enter into opening 4 easily,
and prevents cell 9 once trapped in hollow section 6 from return to
well 2 easily.
[0089] In order to make the wall of opening 4 taper, substrate 1
may be etched with only the etching-accelerator gas. In this case,
as shown in FIG. 22, the diameter of opening 4 at the boundary
against well 2 becomes greater than the diameter defined by resist
mask 12. Therefore, the diameter defined by resist mark 12 is
determined in advance in order to get an optimum taper shape.
[0090] According to Embodiment 1, the relation among the diameters
of openings 4 and 5 and hollow section 6 is described. As shown in
FIG. 3, connecting portion 102, which is a border between opening 4
and hollow section 6, has a diameter smaller than the maximum
diameter of hollow section 6. Connecting portion 103, which is a
border between opening 5 and hollow section 6, has a diameter
smaller than that of connecting portion 102. This arrangement
provides advantages identical to those discussed above.
Exemplary Embodiment 2
[0091] FIG. 23 is a perspective view of a device for measuring an
extracellular potential in accordance with Exemplary Embodiment 2
of the present invention. FIG. 24 is a sectional view of the
device. FIG. 25 is an enlarged sectional view of the device.
Substrate 16 is formed by stacking first silicon layer 17, silicon
dioxide layer 18, and second silicon layer 19, differently from a
device of Embodiment 1. First opening 21, second opening 22, and
hollow section 23 are formed in first silicon layer 17, second
silicon layer 19, and silicon dioxide layer 18, respectively.
[0092] Detecting electrode 24 is formed only on an inner wall of
second opening 22 and a lower portion of hollow section 23, and
leading electrode 25 is formed on a lower surface of substrate 16.
Detecting electrode 24 is electrically connected to leading
electrode 25 around second opening 22.
[0093] An operation of the device discussed above is identical to
that of Embodiment 1, and the description thereof is thus omitted.
Silicon dioxide layer 18 between first silicon layer 17 and second
silicon layer 19 increases electrical insulation between the
layers. Therefore, an electric signal generated by activity of a
cell at second opening 22 can be detected securely by detecting
electrode 24, and the signal does not leak to first opening 21.
[0094] Processes for manufacturing the device in accordance with
Embodiment 2 are described below. A description of processes
identical to those of Embodiment 1 is omitted, and only processes
for forming first opening 21, second opening 22, and hollow section
23 will be described below. The substrate includes the silicon
layer, the silicon dioxide layer, and the silicon layer stacked in
this order, which is available in market as an SOI substrate, and
is not thus explained.
[0095] First, well 20 is formed in first silicon layer 17, and
then, as shown in FIG. 26, resist masks 26 and 27 is provided in
order to form first opening 21 and second opening 22. Next, as
shown in FIG. 27, layers 17 and 19 are dry-etched from a bottom of
well 20 and a lower surface of substrate 16, respectively, so that
respective walls of the openings become perpendicular to the bottom
of well 20, and the openings reach silicon dioxide layer 18. The
substrate is etched, similarly to Embodiment 1, with
etching-accelerator gas for facilitating the etching and
etching-suppressor gas for suppressing the etching. In order to
form openings 21 and 22, layers 17 and 19 are etched until the
openings reach silicon dioxide layer 18. This etching requires no
monitoring of an etching time for obtaining a predetermined
depth.
[0096] Next, the substrate is dipped into solution of HF, which
mainly etches silicon dioxide layer 18 and etches layer 17 and 19
little to form hollow section 23, as shown in FIG. 28. Layer 18 is
etched until hollow section 23 has a necessary lateral diameter.
Then, similarly to Embodiment 1, detecting electrode 24 and leading
electrode 25 are formed. Layer 18 may be etched with plasma using
HF gas, which etches the silicon layers little but etches mainly
silicon dioxide layer 18 similarly to the HF solution. An etching
of Embodiment 1, an etching of excessively long time does not make
the hollow section oval; however, the method of Embodiment 2
overcomes this problem.
[0097] In substrate 16 including two kinds of layers, namely,
silicon and silicon dioxide layers, the depth of second opening 22
and the height of hollow section 23 are determined in advance, thus
allowing the device to be manufactured easily. Silicon dioxide
layer 18 completely isolates first opening 21 electrically from
second opening 22, thus providing a reliable measuring device.
[0098] According to Embodiment 2, substrate 16 includes three
layers, i.e., first silicon layer 17, silicon dioxide layer 18, and
second silicon layer 19. However, the substrate may include four
layers, i.e., a silicon layer, a silicon dioxide layer, a silicon
layer, and a silicon dioxide layer, or more than four layers.
[0099] Substrate 16 is formed by stacking the silicon layer, the
silicon dioxide layer, and the silicon layer in this order.
However, a substrate formed by stacking a silicon dioxide layer, a
silicon layer, and a silicon dioxide layer in this order may
provide the device. Substrate 16 may be made of not only the
combination of silicon and silicon dioxide, but also other
combinations, such as silicon and glass, aluminum and aluminum
oxide, or glass and resin. Substrate 16 may be made of three
materials instead of the two materials, and may include layers of
materials different from each other. Such substrates provide
advantages similar to those discussed above.
[0100] Similarly to Embodiment 1, as shown in FIG. 3, a first
connection section at a border between the first opening and the
hollow section has a diameter smaller than the maximum diameter of
the hollow section, and a second connection section at a border
between the second opening and the hollow section has a diameter
smaller than that of the first connection section. This structure
provides advantages similar to those of Embodiment 2.
Exemplary Embodiment 3
[0101] FIG. 30 is a perspective view of a device for measuring an
extracellular potential in accordance with Exemplary Embodiment 3
of the present invention. FIGS. 31A, 31B and 32 are sectional views
of the device. FIGS. 33 and 34 are enlarged sectional views of the
device. FIG. 35 through FIG. 42 are sectional views of the device
for illustrating a method of manufacturing the device.
[0102] As shown in FIG. 30 through FIG. 32, substrate 28 has a
laminated structure including base 29 made of silicon, intermediate
layer 30 made of silicon dioxide, and thin plate 31 made of
silicon. Base 29 has well 32 therein for accommodating sample
solution including test cells. Well 32 is used for mixing test
cells 37 and culture solution with chemicals. Further, thin plate
31 forming the bottom of well 32 has through-holes 33 therein. Well
32 has pockets 34 formed at holes 33, and thus, a diameter of hole
33 at well 32 is greater than a diameter of hole 33 at a lower
surface of substrate 28.
[0103] Diameters of through-holes 33 and pockets 34 may be
determined according to a size and characteristics of test cell 37.
For the cell 37 having a diameter of 10 .mu.m, the pocket 34 having
a diameter ranging from 10 .mu.m to 20 .mu.m and the hole 33 having
a diameter smaller than 5 .mu.m are suitable.
[0104] According to Embodiment 3, an inner wall of pocket 34 has a
conical shape having its bottom towards well 32.
[0105] Insulator 36 made from silicon dioxide is provided on the
inner wall and the bottom of well 32, the inner wall of
through-hole 33, the inner wall of pocket 34, and the lower surface
of thin plate 31. Detecting electrodes 35 made mainly of gold are
provided on a portion of insulator 36 on the inner wall of hole 32
and the outside of thin plate 31.
[0106] The cell generally has a diameter of 5 .mu.m-20 .mu.m, and
thus, an opening of pocket 34 preferably has a diameter of 10
.mu.m-100 .mu.m, and an opening of hole 33 has a diameter of 1
.mu.m-10 .mu.m. The device discussed above can measure an
extracellular potential, i.e., a physico-chemical change generated
by the cell by the following operation described below with
reference to figures.
[0107] FIG. 32 is a sectional view of well 32 having test cell 37
and culture solution 38 put therein. FIGS. 33 and 34 are enlarged
sectional views of an essential portion including through-hole 33
and pocket 34. As shown in FIG. 32, just after culture solution 38
and test cell 37 are input in well 32, cell 37 floats in solution
38. Well 32 is filled with solution 38 as well as pocket 34 and
hole 33 are filled with solution 38, and then, solution 38
overflows to the lower surface of well 32. As shown in FIG. 33,
this flow allows floating cell 37 to be sucked in pocket 34 by a
pressure of solution 38 in well 32. If the pressure is small,
solution 38 may be sucked with a suction pump from hole 33 to allow
floating cell 37 to be sucked more securely in pocket 34.
[0108] Next, test cell 37 reaching pocket 34 receives a pressure by
suction from hole 33 or by culture solution 38 from well 32, thus
being retained in pocket 34, as shown in FIG. 33. At this moment,
chemicals (not shown) may be doped into culture solution 38 in well
32 to permeate into solution 38. When the chemicals activates test
cell 37 due to reaction by ion-exchange, as shown in FIG. 34, an
electric signal generated in hole 33 changes an electric potential
of a portion of culture solution 38 filled in hole 33. This
electric potential change is detected by detecting electrode 35
contacting solution 38.
[0109] As described above, pocket 34 provided in the bottom of well
32 allows the device in accordance with Embodiment 3 not to require
another well. Test cell 37 and the culture solution can be mixed
with the chemicals in well 32. Well 32, pockets 34 provided in the
bottom, and through-holes 33 are unitarily formed, thus preventing
culture solution 38 from leaking outside well 32 by mistake, and
thus allowing the solution to flow to aperture 33.
[0110] Insulator 36 of silicon dioxide provided on the inner wall
of pocket 34, the inner wall of through-hole 33, the lower surface
of thin plate 31, the bottom, and the inner wall of well 32
electrically insulates detecting electrode 35 from well 32. Since
pocket 34 has the conical shape having its bottom towards well 32,
cell 37 is sucked into pocket 34 and is retained in the pocket
stably, thus preventing cell 37 from staying in aperture 33. For
instance, if test cell 37 has a diameter of 10 .mu.m, the diameter
of pocket 34 at well 32, namely, the bottom of the conical shape is
determined to be less than 20 .mu.m, thus plural cells 37 not to
enter into pocket 34 at once. Through-hole 33 having a diameter
less than 5 .mu.m does not allow cell 37 to pass through hole
33.
[0111] As discussed above, test cell 37 can be securely retained in
pocket 34 during measuring. A portion of culture solution 38 in
hole 33 is electrically insulated from a portion of solution 38 in
well 32, thus preventing the electric signal generated by activity
of test cell 37 from leaking to the portion of solution 38 in well
32. Therefore, the signal is detected by detecting electrode 35
provided on hole 33. Insulator 36 is necessary when a surface layer
of the silicon substrate has a small resistivity. Alternatively,
insulator 36 is necessary when the electric signal generated in
hole 33 is too weak to be measured due to a little leak of the
electric signal to well 32.
[0112] Therefore, if the silicon substrate has a large surface
resistivity, test cell 37 that is retained assures enough electric
insulation. Therefore, when an extracellular potential is large
enough not to be influenced by a little leakage of the electric
signal, insulator 36 is not necessarily required.
[0113] Next, a method of manufacturing the device in accordance
with Embodiment 3 will be described below with reference to FIG. 35
to FIG. 42. First, as shown in FIG. 35, substrate 38 including base
29 made of silicon, intermediate layer 30 made of silicon dioxide,
and thin plate 31 made of silicon is prepared. Resist mask 39 is
provided on the lower surface of thin plate 31. Substrate 28 may be
an SOI substrate, which is often used for manufacturing
semiconductor devices. The SOI substrate is available in market,
and thus, a method for manufacturing the substrate is not
described.
[0114] Then, thin plate 31 is dry-etched to form through-hole 33
having a predetermined depth. FIG. 36 is an enlarged of portion A
in FIG. 35. It is important that the plate is preferably dry-etched
with etching-accelerator gas for facilitating the etching and
etching-suppressor gas for suppressing the etching, similarly to
Embodiment 1. These gases allow through-hole 33 to be formed only
beneath resist mark 39, as shown in FIG. 36.
[0115] While substrate 28 is dry-etched with the
etching-accelerator gas and the etching-suppressor gas used
alternately, a high frequency is applied to substrate 28, and an
inductive-coupling method with an external coil is used for the
etching. The high frequency generates a negative bias potential in
substrate 28 and makes positive ions in plasma, such as
SF.sub.5.sup.+ or CF.sub.3.sup.+ collide with substrate 28, thus
allows substrate 28 to be etched perpendicularly to the bottom.
[0116] The dry-etching may be suppressed by stopping the applying
of the high frequency to substrate 28. The bias potential is
stopped, and CF.sup.+, material of a protective film, is not
deflected. As a result, the protective film is formed uniformly on
the walls of the through-holes in substrate 28.
[0117] The method described above is effectively applicable to
forming an opening perpendicular to the bottom of the substrate by
manufacturing methods described in Embodiments 1 and 2.
[0118] Next, as shown in FIG. 37, thin plate 31 is dry-etched until
the hole reaches intermediate layer 30. In this process, a
concentration of the etching-accelerator gas is gradually increased
according to the progress of the etching toward the bottom of well
32. Alternatively, a time of the etching with the
etching-accelerator gas is gradually increased. In other words,
when facilitating of the dry-etching and suppressing of the
dry-etching are alternately repeated, the ratio of a time of the
facilitating to a time of the suppressing is gradually
increased.
[0119] This operation allows the hole 33 to flaring toward well 32,
as shown in FIG. 37, thus allowing through-hole 33 to communicate
with pocket 34 flaring toward well 32. When the dry-etching is
finished, i.e. when hole 33 reaches intermediate layer 30, the
dry-etching is not necessarily stopped immediately due to
intermediate layer 30 made from silicon dioxide since the etching
gas does not etch intermediate layer 30 immediately.
[0120] The etching-accelerator gas of SF.sub.6 cannot easily
dry-etch intermediate layer 30 made of silicon dioxide to remove
the intermediate layer since a ratio of an etching rate for silicon
to that for silicon dioxide is more than ten. Therefore, even if
the dry-etching continues for a while after the hole reaches the
silicon dioxide layer, the dry-etching can hardly remove layer 30,
thus forming pocket 34 accurately and easily.
[0121] Then, as shown in FIG. 38, resist mask 40 is provided on
base 29 by a photo-lithography method. Then, as shown in FIG. 39,
base 29 is etched until well 5 reaches intermediate layer 30. At
this process, the etching-accelerator gas and the
etching-suppressor gas may be used, as previously discussed, for
providing wells 32 at a high density. However, if the high density
is not needed, a wet-etching using TMAH or KOH is acceptable.
[0122] Next, as shown in FIG. 40, a portion of intermediate layer
30 made of silicon dioxide exposed from the bottom of well 32 is
removed by a wet-etching using HF or a dry-etching with CF.sub.4
gas.
[0123] Then, as shown in FIG. 41, a silicon-dioxide layer is formed
on the surface of the silicon substrate, which includes base 29 and
thin plate 31, by a thermal oxidation method. This process provides
insulating layer 36 made from silicon dioxide on the inner wall and
the bottom of well 32, the inner wall of pocket 34, the inner wall
of hole 33, and the lower surface of thin plate 31.
[0124] Next, as shown in FIG. 42, detecting electrode 35 is formed
on the lower surface of thin plate 31 by vapor-depositing or
sputtering gold. Thus, electrode 35 is formed not only on the lower
surface of plate 31 but also on the inner wall of through-hole 33.
Electrode 35 is made of a material so as not to react on culture
solution 38. The material may be preferably selected from gold,
platinum, silver, silver chloride, and aluminum appropriately
according to a type of the sample solution.
[0125] The method in accordance with Embodiment 3 described above
provides the device having through-holes 33 in thin plate 31 and
conical pockets 34 communicating with holes 33 to well 32
accurately and easily by a one-time etching.
[0126] In the method of Embodiment 3, it is not necessary that the
substrate is etched with two kinds of resist masks by the
photolithography method from the well. This method allows
through-holes and pockets to be formed accurately in the bottom of
the well even if the substrate has bumps and dips therein. Even the
substrate having such rough surface may have the through-holes and
the pockets in the bottom of the well accurately without using a
spray-coating device which coat the rough surface uniformly with a
resist mask, or using a projection or a stepper forming a highly
accurate pattern onto the resist mask by exposure to light with non
contact between a photo mask and the substrate.
Exemplary Embodiment 4
[0127] FIG. 43 is a sectional view of a device for measuring an
extracellular potential in accordance with Exemplary Embodiment 5
of the present invention. FIGS. 44 and 45 are enlarged sectional
views of essential portions of the device.
[0128] The device in accordance with Embodiment 5 has a structure
basically identical to that of Embodiment 3, and thus, similar
elements are not described.
[0129] FIG. 43 is a sectional view of the device in accordance with
Embodiment 5. Pocket 47 formed in thin plate 44 has a hemisphere
shape, which retains test cell 37 more closely thereto, so that a
change of an electrical potential of culture solution 38 in
through-hole 46 can be detected more easily.
[0130] A method of manufacturing the device will be described
below. The method in accordance with Embodiment 4 is similar to
that of Embodiment 3. According to Embodiment 4, through-hole 46
and pocket 47 is formed in thin plate 44 by a method different from
that of Embodiment 3. The different method will be described with
reference to FIGS. 44 and 45.
[0131] As shown in FIG. 44, resist mask 50 is formed on thin plate
44 while intermediate layer 43 and thin plate 44 are attached to
each other. Then, thin plate 44 is dry-etched with
etching-accelerator gas for facilitating the etching and
etching-suppressor gas for suppressing the etching up to a
predetermined depth to form through-hole 46. The predetermined
depth is determined to prevent the etching from reaching
intermediate layer 43 made of silicon dioxide, and determined to be
an optimum depth in response to a size and a shape of pocket
47.
[0132] During this etching, thin plate 44 is etched with the
etching-accelerator gas, and then has a protective film (not shown)
formed thereon with the etching-suppressor gas. These processes for
dry-etching thin plate 44 are repeated perpendicularly to resist
mask 50 and only under an opening of resist mask 50. These
processes terminate by dry-etching thin plate 44 with the
etching-accelerator gas. This operation removes the protective film
formed by the etching-suppressor gas from the bottom of the etched
place.
[0133] Next, as shown in FIG. 45, pocket 47 is formed by
dry-etching with XeF.sub.2 gas. The dry-etching progresses from the
bottom where silicon is exposed, and corroded area becomes greater
as the etching progresses toward well 45. The inner wall of
through-hole 46 has the protective film formed thereon by the
etching-suppressor gas, so that the wall of hole 46 is not
dry-etched by the XeF.sub.2 gas. Pocket 47 thus has a hemisphere
shape, as shown in FIG. 45 shows. After these processes discussed
above, the substrate undergoes the processes shown in FIG. 38
through FIG. 42 similarly to that of Embodiment 3, thereby
providing the device for measuring an extracellular potential.
Exemplary Embodiment 5
[0134] According to Exemplary Embodiment 5, another method for
forming through-hole 33 and 46 and pocket 34 and 47 described in
Embodiments 3 and 4, respectively, will be described.
[0135] The method of forming the through-hole different from the
methods of Embodiments 3 and 4 will be described hereinafter with
reference to FIG. 46 through FIG. 48.
[0136] FIG. 46 is a sectional view of a measuring device in
accordance with Embodiment 5 for illustrating a method of
manufacturing the device. Intermediate layer 51 made of silicon
dioxide and thin plate 52 made of silicon are stacked on each
other. Then, resist mask 53 is provided on thin plate 52, and
through-hole 56 is formed by dry-etching a substrate with
etching-accelerator gas for facilitating the etching and
etching-suppressor gas for suppressing the etching. Thin plate 52
is continued to etch until hole 56 reaches intermediate layer
51.
[0137] Thin plate is still continued to etch after hole 56 reaches
intermediate layer 51 which is made of insulator and has a
resistance smaller than thin plate 52 made of silicon. As shown in
FIG. 47, excessive etching makes etching ions 54, such as
SF.sub.5.sup.+, stay on the surface of layer 51 when the
etching-accelerator gas is used for the dry-etching, so that
etching ions 55 supplied from plasma deflects along an arrow shown
in FIG. 47.
[0138] As a result, the vicinity of the wall of intermediate layer
51 is locally dry-etched, and through-hole 56 flares toward the
well, namely, pocket 57 is formed, as shown in FIG. 48.
[0139] According to an experiment, thin plate 52 is continued to
dry-etch after hole 56 having a diameter of 3 .mu.m reaches
intermediate layer 51, thereby providing pocket 57 having a maximum
diameter of 10 .mu.m.
INDUSTRIAL APPLICABILITY
[0140] A device for measuring an extracellular electric potential
according to the present invention allows a test cell to enter in a
hollow section of the device. Once the test cell enters in the
hollow section, the cell is trapped therein securely. The device
thus can detect an electric signal generated by activities of the
cell without fail.
* * * * *